1. Field of the Invention
This invention relates to a novel fungicidal saponin and its isolation from cayenne pepper or other capsicum species fruit. This compound shows potential for improving crop resistance to a variety of aflatoxin-producing fungi and also as a pharmaceutical against human and animal fungal-induced diseases.
2. Description of the Prior Art
The identification of novel antifungal agents has been a continuing challenge. Due to the occurrence of new disease epidemics in agricultural crops and as a result of the growing resistance to antimicrobial drugs, there is an ongoing search for new antifungal compounds having unique structures.
The need for novel antifungal agents is significant, and is especially critical in the medical field. Immunocompromised patients provide perhaps the greatest challenge to modern health care delivery. During the last three decades there has been a dramatic increase in the frequency of fungal infections in these patients (Herbrecht, R., Eur. J. Haematol., 1996, 56:12-17; Cox, G. et al., Curr. Opin. Infect. Dis., 1993, 6:422-426; Fox, J. L., ASM News, 1993, 59:515-518). Deep-seated mycoses are increasingly observed in patients undergoing organ transplants and in patients receiving aggressive cancer chemotherapy (Alexander, B. D. et al., Drugs, 1997, 54:657-678). The most common pathogens associated with invasive fungal infections are the opportunistic yeast, Candida albicans, and the filamentous fungus, Aspergillus fumigatus (Bow, E. J., Br. J. Haematol., 1998, 101:1-4; Wamock, D. W., J. Antimicrob. Chemother., 1998, 41:95-105). There are an estimated 200,000 patients per year who acquire nosocomial fungal infections (Beck-Sague, C. M. et al., J. Infect. Dis., 1993, 167:1247-1251); bloodstream fungal infections have a mean mortality rate of 55 w. Also adding to the increase in the numbers of fungal infections is the emergence of Acquired Immunodeficiency Syndrom (AIDS) where virtually all patients become affected with some form of mycoses (Alexander, B. D. et al., Drugs, 1997, 54:657-678; HIV/AIDS Surveillance Report, 1996, 7(2), Year-End Edition; Hood, S. et al., J. Antimicrob. Chemother., 1996, 37:71-85). The most common organisms encountered in these patients are Cryptococcus neoformans, Pneumocystis carinii, and C. albicans (HIV/AIDS Surveillance Report, 1996, 7(2), Year-End Edition; Polis, M. A. et al., AIDS: Biology, Diagnosis, Treatment and Prevention, fourth edition, 1997). New opportunistic fungal pathogens such as Penicillium marneffei, C. krusei, C. glabrata, Histoplasma capsulatum, and Coccidioides immitis (HIV/AIDS Surveillance Report, 1996, 7(2), Year-End Edition; Ampel, N. M., Emerging Infectious Diseases, 1996, 2:109-116) are also being reported with regularity in immunocompromised patients throughout the world.
Currently available drugs for the treatment of fungal infections include amphotericin B (a macrolide polyene), which interacts with fungal membrane sterols, flucytosine (a fluoropyrimidine), which interferes with fungal protein and DNA biosynthesis, and a variety of azoles (e.g. ketoconazole, itraconazole, and fluconazole) that inhibit fungal membrane-sterol biosynthesis (Alexander, B. D. et al., Drugs, 1997, 54:657-678). Even though amphotericin B has a broad range of activity and is viewed as the "gold standard" of antifungal therapy, its use is limited due to infusion-related reactions and nephrotoxicity (Alexander, B. D. et al., Drugs, 1997, 54:657-678; Wamock, D. W., J. Antimicrob. Chemother., 1998, 41:95-105). Flucytosine usage is also limited due to the development of resistance and its narrow spectrum of activity. The widespread use of azoles is causing the emergence of clinically-resistant strains of Candida spp. (Alexander, B. D. et al., Drugs, 1997, 54:657-678; Boschman, C. R. et al., Antimicrob. Agents Chemother., 1998, 42:734-738). Although advances in the formulation of amphotericin B have decreased its nephrotoxicity (Graybill, J. R., Clin. Infect. Dis., 1996, 22(Suppl. 2):S166-S178) and new classes of agents are in various stages of clinical development, the impact of fungal infections in the clinical management of patients underscores the clear need for new antifungals.
Historically, the screening of soil microorganisms and extracts obtained from terrestrial plants and animals has yielded novel natural products which themselves, or through chemical modification and synthesis, have been a rich source of bioactive agents for the treatment of plant, animal and human diseases (Gullo, V. P., The discovery of natural products with therapeutic potential, Butterworth-Heinemann, Boston, 1994; Shu, Y.-Z., J. Nat. Prod., 1998, 61:1053-1071). A review of the 520 newly-approved drugs reported between 1983 and 1994 indicates that 157 (30%) are unmodified natural products or semi-synthetic analogs (Cragg, G. M. et al., J. Nat. Prod., 1997, 60:52-60).
The screening of natural sources such as microbial fermentations and plant extracts has led to the discovery of many clinically useful drugs that play a major role not only in the treatment of microbial infections, but also in the treatment of many human and plant diseases. The increasing clinical importance of emerging pathogens as well as drug-resistant fungi has lent additional urgency to identify novel, active compounds. The most promising lead antifungal compounds are those derived from natural products. The lipopeptides are potent broad-spectrum antifungal agents that inhibit the synthesis of the fungal cell-wall polymer, (Gullo, V. P., The discovery of natural products with therapeutic potential, Butterworth-Heinemann, Boston, 1994; Cragg, G. M. et al., J. Nat. Prod., 1997, 60:52-60) .beta.-D-glucan (Denning, D. W., J. Antimicrob. Chemother., 1997, 40:611-614). The pradimicins are a group of benzonaphthacene quinones which posses a broad spectrum of activity and bind to the mannoproteins of the fungal cell membrane (Oki, T. et al., J. Antibiot., 1998, 41:1701-1704). The sordarins are tetracyclic diterpene glycosides, which have a broad spectrum of antifungal activity and inhibit the elongation step of fungal protein synthesis (Drugs Future, 1997, 22:1221-1225).
Another class of antifungal agents are the saponins, which are glycosides consisting of one or more sugars linked to a steroid or triterpene core. They are noted for their detergent properties and some have been found to be microbicidal. Some are antiviral (Sindambiwe, J. B. et al., J. Nat. Prod., 1998; 61:585-590; Simoes, C. M. O. et al., Planta. Med., 1990, 56:652-653; Simoes, C. M. O. et al., Phytother. Res., 1999, 13:323-328) and appear to inhibit viral DNA and capsid protein syntheses (Simoes, C. M. O. et al., Phytother. Res., 1999, 13:323-328), while others have antifungal properties. Extracts of Eriocephalus africanus L., Felicia erigeroides DC, and Helichrysum crispum (L.) D. Don, inhibited Candida albicans growth (Salie, F. et al., J. Ethanopharmacol., 1996, 52:27-33). Saponins from the tubers of Cyclamen coum Miller inhibited the growth of several Candida species and Cryptococcus neoformans (Calis, I. et al., Planta. Med., 1997, 63:166-170). Triterpenoid saponins have been shown to exhibit a wide spectrum of activity against yeast as well as the dermatophytes, Trichophyton rubrum and T. mentagrophytes (Favel, A. et al., Planta. Med., 1994, 60:50-53). Recently, a jujubogenin saponin isolated from Colubrina retusa, a rhamnaceous plant growing in Venezuela (Li, X. C. et al., J. Nat. Prod., 1999, 62:672-677), was shown to be moderately effective against C. albicans, Cryptococcus neoformans, and A. fumigatus (MICs, 50 .mu.g/ml). Oats contain two families of saponins, the triterpenoid avenacins and the steroidal avenacosides (Osborn, A. E. et al., The Plant Cell, 1996, 8:1821-1831).
Fenugreek produces a steroid saponin and the corresponding aglycone that becomes inhibitory against fungi such as Candida albicans after treatment with .beta.-glucosidase (Sauvaire, Y. et al., Saponins Used in Foods and Agriculture, Plenum Press, New York, 1996, pp. 37-46). Stems of Colubrina retusa contain several jujubogenin saponins with no to moderate antifungal properties. The most active compound showed growth inhibitory effects (MICs of .about.50 .mu.g/mL) against C. albicans, Cryptococcus neoformans, and A. fumigatus while the other two jujubogenins showed little or no activity (Li, X. et al., J. Nat. Prod., 1999, 62:674-677). Another saponin family, the spirostanol sapogenins from the bulbs of leak (Allium porrum), are antifungal. Four of the eight members of this family inhibited Fusarium culmorum growth with IC.sub.50 values of 30-35 .mu.g/mL (Carotenuto, A. et al., Phytochem., 1999, 51:1077-1082). The salzmannianosides are two saponins isolated from Serjania salzmanniana (Ekabo, O. et al., J. Nat. Prod., 1996, 59:431-435) and displayed activity against Candida albicans and Cryptococcus neoformans at 16 and 8 .mu.g/mL, respectively. Three triterpene saponins, cyclainorin, deglucocyclamin, and from Cyclamen mirabile and Cyclamen coum were shown to have inhibitory properties against a number of Candida species (Cali&Ocirc ;, I. et al., J. Nat. Prod., 1997, 60:315-31). Not all saponins have antifungal properties.
As described hereafter, our search for antifungal compounds from higher plant species has included investigation of the genus Capsicum. Capsicum is a member of the Solanaceae, a large tropical plant family (Eshbaugh, W. H., New Crops, Wiley, New York, 1993, pp. 132-139). However, only five species of peppers have been domesticated; C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens. They are noted for the pungency and medicinal properties of their fruit. C. frutescens, commonly known as cayenne pepper, is also used in the Tabasco.RTM. pepper products.
Peppers have been used for many centuries to inhibit food spoilage (Billing, J. et al., Q. Rev. Biol., 1998, 73:2-47). For example, the Mayan pharmacopoeia describes the use of the tissues of certain Capsicum species in herbal remedies for a variety of ailments of microbial origin (Cichewicz, R. H. et al., J. Ethanopharm., 1996, 52:61-70). To date, only a few bioactive compounds have been isolated from this pepper family. The most commonly-known compounds belong to the capsaicinoid group, of which capsaicin (n-vanillyl-8-methyl-6-(E)-nonamide) is the predominant species. Capsaicin has been studied extensively and has demonstrated a high degree of biological activity affecting nervous, cardiovascular, and digestible systems (Virus, R. M. et al., Life Sci., 1979, 25:1273-1284; Monsereenusom et al., CRC Critical Reviews in Toxicology, 1982, 10:321-339; Surh, Y. J. et al., Life Sci., 1995, 56:1845-1855). Presently, two prescription drugs are based on capsaicin. Zostrix.RTM. (Genderm) is used for the treatment of shingles and arthritis while Axsain.RTM. (GalenPharma) is used for relief of neuralgias, diabetic neuropathy, and postsurgical pain.
Most of the compounds isolated from this genus have been obtained from C. annuum (bell pepper). One such compound is a 75 amino-acid polypeptide named J1 (Meyer, B. et al., Plant Physiol., 1996, 112:615-622) which acts as a fungistatic agent against Fusarium oxysporum and Botrytis cinerea. Bell pepper stems produce a 34 kDa .beta.-1,3-glucanase, which has been shown to inhibit hyphal growth of Phytophthora capsici (Kim, Y. J. et al., Physiol. Mol. Plant Pathol., 1997, 50:103-105) and to act synergistically with a pepper chitinase in inhibiting the growth of F. oxysporum.
C. annuum roots and seeds produce steroidal glycosides, named capsicosides A-D (Yahara, S. et al., Phytochem., 1994, 37:831-835), having a wide range of biological properties such as froth formation, hemolytic activity, complexing with cholesterol, antitumor, antitussive, and platelet-aggregation inhibitory properties (Mimaki, Y. et al., Saponins Used in Traditional and Modern Medicine, Plenum Press, New York, 1996, pp. 101-110; Back, K. et al., Plant Cell. Physiol., 1998, 39:899-904). To date, none of the Capsicum species has been identified as producing saponins with antifungal properties.